In recent years, the use of recombinant DNA technology and the systematic analysis of biological data have increased considerably, yielding Metabolic Pathway Engineering (MPE), which is defined as the modification and/or introduction of new biochemical reactions for the direct improvement of cellular properties through recombinant DNA technology (Stephanopoulos, 1999; Bailey, 1991). Specifically, new strains are now being developed through MPE that have the property of being able to grow in mineral media and to produce primarily a single microbial metabolite—for example, only one lactate isomer (Bai et al. 2003; Dien et al., 2002; Zhou et al. 2003a and 2003b; Zhu and Shimizu 2004; Zhou et al., 2006a; Zhou et al. 2006b; Zhou et al., 2005).
Lactic Acid.
In the chemical industry, especially in the manufacturing of raw materials for the production of plastics of biological origin, the biotechnological production of lactic acid has attracted a large amount of interest recently, as this compound offers a sustainable alternative for the manufacturing of high-quality biodegradable plastics known by the generic name of polylactates (PLAs); examples include polylactate and ethyl-lactate (Dien et al., 2002; Skory, 2003). The synthesis of biodegradable PLAs requires the separate production of the D and L lactate isomers. In addition, the physical and biodegradative properties of PLA depend on the proportion of the D and L forms used in the synthesis of the polymer. Lactate can be produced by microbial fermentation or by chemical synthesis (Narayanan et al., 2004). The most commonly used chemical process is the hydrolysis of lactonitrile with strong acids; however, there are other chemical routes (John et al., 2007), such as the oxidation of propylene glycol, the reaction of acetaldehyde with carbon monoxide and water at high temperatures and the hydrolysis of chloropropionic acid, among others. All of these routes yield a mixture of D and L isomers as a final product and depend on raw materials derived from petroleum, which makes these production processes less sustainable. In contrast, the biotechnological production of lactic acid has several advantages over chemical synthesis: 1) the low cost of the substrates, 2) the low production temperature, 3) the low energy consumption and 4) the specificity for the desired stereoisomer. The lactate is produced through a process of microbial fermentation of culture media with an easily assimilated carbon source, such as glucose.
Ethanol
One of the most difficult challenges in the present search for substitutes for fuels derived from petroleum is the identification of possible alternative liquid fuels.
The production of ethanol from biomass is one of the few currently viable options (Mielenz, 2001). Several technologies are in the growth stage; a large variety of raw materials can be used; and the ethanol produced is a valuable and versatile compound, as it can be used as an oxygenating agent, fuel or solvent or be transformed, using established technologies, into other fuels (e.g., biodiesel) (Bungay, 2004).
Ethanol can be used for many applications. The primary application discussed in this document is as a liquid fuel that will oxygenate, substitute for or complement fossil fuels that are currently used in internal combustion engines. Other applications of ethanol include its use as a fuel in industrial boilers, lamps, furnaces, turbines, among others.
When compared in volumetric terms, the energetic content of ethanol is approximately two-thirds of that stored in gasoline or diesel. However, ethanol has a high octane value, which causes the engines that use gasoline-ethanol mixtures to have a better efficiency. Mixtures that contain up to 22% (v/v) ethanol can be used successfully in current gasoline engines, that is, without the need of modifying these internal combustion engines.
Another alternative use of ethanol is as an oxygenating agent. To improve combustion and to reduce the levels of carbon monoxide produced, fuels need to elevate their octane value without using lead. To that end, alcohols and esters have been used. Currently, in Mexico, tert-butyl ethers are used, of which methyl tert-butyl ether (MTBE) is the most commonly used. However, it is known today that these compounds can accumulate in groundwater, are resistant to chemical and biological degradation and are carcinogenic to humans in parts per million concentrations. In several states, such as California, their use has been prohibited.
Traditionally, ethanol is obtained through the fermentation of glucose or sucrose, which are obtained from corn starch and cane sugar, respectively. This fermentation is conducted using ethanol-generating organisms, such as Saccharomyces cerevisiae. This organism is traditionally used for the production of ethanol from glucose, which is generated from the hydrolysis of grain starch and sucrose obtained from cane sugar or sugar beet. This microorganism does not have the ability to metabolize the five-carbon sugars, known as pentoses that are abundantly found in hydrolyzed vegetable material (Hahn-Hagerdal of al., 1993). Another ethanol-generating organism is Zymomonas mobilis, a Gram-negative bacterium, which has the native ability to produce a good yield of ethanol due to its metabolic characteristics. Among these characteristics are two very efficient enzymatic activities, those of pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (Adh), which convert pyruvate into acetaldehyde and ethanol, respectively. However, as also occurs with S. cerevisiae, Z. mobilis is limited in the sugars that it can metabolize. This organism can only use sucrose, glucose and fructose, and it does not use xylose, other pentoses or other disaccharides.
Carbon Sources
Glucose
Cellulose is the greatest component of lignocellulose (20-50%). It is a linear polymer composed of dextrose subunits (D-glucose) that are joined by glycosidic bonds β-(1-4), and due to its structural conformation, it is highly resistant to hydrolysis. To take advantage of cellulose, it is necessary to hydrolyze it with cellulases. The hydrolysis of cellulose yields glucose, which is fermentable by the strains mentioned in the present invention. Glucose is primarily obtained from the hydrolysis of starch
Xylose and Other Monomers
In contrast to cellulose, hemicellulose is not chemically homogeneous, as it is a heterogeneous polysaccharide that contains hexose monomers (glucose, mannose and galactose), pentose monomers (xylose and arabinose) and several acids (acetic acid and glucuronic acid). This composition increases the difficulty of the bioconversion of hemicellulose to fermentation products that are of interest for industrial use. In addition, hemicellulose is the second most common polysaccharide in nature, as it represents 20-35% of the cell mass of lignocellulose. The proportions of pentoses and hexoses in hemicellulose are 85 and 15%, respectively, where xylose is the most abundant, followed by glucose and arabinose (75, 15 and 10%, respectively) (Saha, 2003). Hemicellulose can be converted into monomeric sugars through the use of hydrolysis at temperatures below 200° C. using low acid concentrations, although there are several hydrolysis methods: physical, physicochemical, chemical and/or biological (Sun et al., 2002).
Thus, it can be concluded that, excepting glucose, xylose is the most abundant monosaccharide in nature and is generally found polymerized in the hemicellulose fraction of the vegetable tissue. However, the variety of microorganisms that metabolize both pentoses and hexoses is very limited. Furthermore, there are no wild microorganisms that can efficiently catabolize pentoses or mixtures of pentoses and hexoses through fermentation processes into products of industrial interest at high yields (Hernández-Montalvo et al., 2001).
Therefore, the conversion of lignocellulose materials has serious limiting factors, as these materials are composed of sugar polymers, primarily glucose and xylose; xylose is a pentose that is not fermentable by most of the wild or genetically modified microorganisms used in industry, such as Saccharomyces cerevisiae, Corynebacterium glutamicum, certain lactobacilli, Zymomonas mobilis or Bacillus subtillis (Dien et al., 2001). Another disadvantage for the industrial use of lignocellulose materials is that the majority of microorganisms used to this end, such as lactobacilli, require complex culture media, thus increasing the costs of production because of the need for nutrients, product purification, etc. In addition, in the case of lactic acid, most of the microorganisms synthesize only the D-lactic isomer or a mixture of D and L-lactic.
Lactose
Lactose is a disaccharide made up of glucose and galactose molecules joined by a beta 1-4 link. This disaccharide is found in mammalian milk, and it is common to find it in whey as an agro-industrial residue obtained in cheese production.
Escherichia coli 
Among the microorganisms used industrially for the production of D-lactate, species from the genera Lactobacillus, Rhizopus and Escherichia are most commonly used. Of these microorganisms, Escherichia coli have several advantageous characteristics as the base microorganism for the development of strains and for the production of biotechnology products. Among these characteristics are the following: it grows rapidly under aerobic or anaerobic conditions, its complete genome is known, methodologies are available to modify its genome, and it can metabolize both hexoses and pentoses, as well as disaccharides and a wide variety of other sugars and carbon sources, using only mineral salts as nutrients. For this reason, the strategies of metabolic engineering propose changes in the fermentation pathways to modify the balance of carbon toward the desired product, maintaining the redox balance and preventing the formation of subproducts, with the goal of improving the accumulation of a single fermentation product. For example, if the end product is lactic acid (Zhu et al., 2007), a homolactic microorganism is obtained, whereas, if only ethanol is produced, the microorganism is homoethanologenic (Zhou et al., 2008).
According to the functional metabolic network of E. coli in fermentation conditions, for each mole of glucose (Glc) that is catabolized to pyruvate, two moles of ATP are obtained. If half of the pyruvate generated is converted into acetic acid, the yield increases to 3 molATP/molGlc. However, in the case of xylose (Xyl), the yield is only 0.67 molATP/molXyl when E. coli catabolizes this sugar into pyruvate. This value is so low that the enzymes pyruvate formate lyase (Pfl) and acetate kinase (Ack) are essential in the growth of E. coli from xylose in fermentation conditions, as the conversion of one mole of pyruvate into acetyl-CoA and, in turn, into acetate generates one extra mole of ATP, increasing the yield of ATP to 1.5 molATP/molXyl. As a consequence, the E. coli W3110 strains without pflB cannot grow in pentose, as they only yield 0.67 molATP/molXyl. The insufficiency of ATP was confirmed by inactivating the acetate kinase (ack) gene in E. coli W3110. This mutant was incapable of growing in the minimal media supplemented with xylose in anaerobic conditions, verifying the need for the ATP produced by Ack (Hasona et al., 2004). For glucose, the transport and phosphorylation is carried out by the PTS system, with an equivalent cost of ATP. In contrast, for xylose, the cell spends two molecules of ATP, one for the transport (high-affinity ABC transporter) and the second for phosphorylation (Lin, 1996; Linton and Higgins 1998). In arabinose, the internalization of the pentose in the cell is carried out by symport (arabinose/H+) through AraE, a low- and high-affinity transporter. This approach conserves one molecule of ATP spent in the transport of pentoses through the ABC transporter, and both mutants (pfl and ack) grow in arabinose (Hasona et al., 2004).
The Use of E. coli in the Production of Lactic Acid
For the production of lactic acid, E. coli has a gene that codes for an enzyme vital to lactate production, lactate dehydrogenase (IdhA), which is expressed in anaerobic conditions (Zhou et al., 2003a). However, when grown in the presence of glucose or xylose, E. coli is heterofermentative, yielding acetic, formic, lactic and succinic acids, in addition to ethanol, hydrogen and carbon dioxide (Bock and Sawers 1996). Through MPE techniques, E. coli strains have been modified by the blockade of pathways that compete for pyruvate availability to induce the microorganism to become homofermentative and mostly produce D-lactate (U.S. Patent Application No US2007/0037265) from pyruvate; however, those strains were modified to use only glucose as a carbon source and to produce D-lactate, with high conversion yields. In contrast, there are reports that detail the inability of E. coli strains that produce D-lactate to grow using xylose as the main source of carbon, due to the low yield of ATP that is obtained with this sugar (Hasona et al., 2004).
The most commonly used strategy for the generation of E. coli strains that produce high optical purity D-lactate consists of suppressing the gene that codes for the pyruvate formate lyase activating enzyme (pflB) (Zhou et al., 2003a and 2003b; Zhu and Shimizu 2004; Zhou et al. 2006a; Zhou et al., 2006b; Zhou et al., 2005). This strategy has yielded conversion efficiencies of the carbon source into D-lactate above the theoretical 95% value (Zhou et al., 2003a and 2003b) but has restricted the industrial process to use glucose as the only carbon source. Another disadvantage comes as a response to a low availability of acetyl-CoA (a key metabolite in the contribution of carbon backbone to cell mass), yielding strains with a very low or null growth rate in anaerobic growth conditions or with glucose as the only carbon source. Typically, these strains are incapable of growing unless the media is supplemented with acetate (Zhou et al., 2003a), driving up the price of the culture media and/or complicating the industrial process.
The Use of E. coli in the Production of Organic Acids and Ethanol
In contrast, E. coli has a pathway to produce other compounds of industrial interest, such as ethanol, in a natural fashion. However, the amount of alcohol that is produced in this manner is very low. In addition to the product of interest, a mixture of other fermentation products is produced, among which are acetic, formic, succinic and lactic acids (Gonzalez et al., 2002; Dien et al., 2003; Lawford and Rousseau 1996; Lawford and Rousseau, 1997); thus, the microorganism is heterofermentative. With the use of Metabolic Pathway Engineering (MPE), the flow of carbon has been redirected to a heterologous ethanol production pathway in E. coli, yielding strains with a different genetic background from those reported in the present invention that are capable of providing good yields in the fermentation of glucose or xylose to ethanol (Otha et al., 1991).